| Qinghai Lake (also named as Blue Lake) is the largest inland saline lake in China, as a National Nature Reserve, which has been listed in the International Important Wetland Protectorate. It locates on the northeastern Tibetan Plateau at an altitude of3194m, and covers an area of4282.3km. A great variety of microorganisms often inhabite inside water and soil of Qinghai Lake, such as halophile, alkaliphile and psychrophile, that have typical physiological and biochemical characteristics and environmental adaptability. Halophiles, as a kind of new extremophiles, have attracted increasing attentions for its extensive application in recent years. Numerous recent studies were not simply just focused on the fundamental research but on the application research, including osmotic balance mechanism, halocin, compatible solutes, bacteriorhodopsin, bioelectronics, bio-medicine and biotechnological exploitation of these organisms. Based on the advantage of ecological plateau in Qinghai Lake, the diversity, distribution and population evolution of halophiles within the lake remains unknown. Therefore, these fundamental studies are of great significance centering on the halophilic properties, species diversity, systematic evolution and synthesis mechanism of compatible solutes ectoine. In addition, the regional diversity of species has more important practical significance in formulating the local policy of nature conservation, and implementing the biodiversity protection and application of biological resources.In this study, a systematically critical review was given, including living environment, basic classification and osmotic regulating mechanism of halophilic microorganisms. Then the biosynthesis, catabolism and transcriptional regulation mechanism of ectoine, and the industrial production strains and quantitative production strategy were emphatically discussed. Subsequently, this study mainly elaborated on the existence situation and dominant population of halophiles isolated from Qinghai Lake, and the accumulation and influencing factors of intracellular ectoine, and also the structure characteristics and regulation mode of the ect operon in ectoine biosynthesis. Specific results were as follows:1) In order to obtain completely halophilic germplasm resources, and construct phyletic evolution tree of halophilic bacterium and understand evolutional orientation,35bacterial strains were isolated from different water samples in Qinghai Lake using an Oesterhelt-Stoeckenius medium (OSM). The majority of halophiles in this lake were moderate halophiles, accounting for about62.9%of total bacteria, followed by the slight halophiles (22.9%) whereas the halotolerant and nonhalophile represented11.4%and2.9%of total bacteria, respectively. We investigated and analyzed the population diversity and evolutionary relationship based on the16S rRNA sequences. A total of35bacterial strains were sequenced, and homology analysis showed that a majority was affiliated with γ-Proteobacteria and Bacilli, which accounted for68.6%and17.1%of total bacteria, respectively, whereas Actinobacteridae and a-Proteobacteria represented a small portion of total bacteria. Based on phylogenetics,35strains belonged to14different genera. Ten strains of Halomonas in the Oceanospirillales were the most dominant species, whereas four strains of Marinomonas represented a minor species component. Genus of Halomonas, as a model organism, has been widely used in the study of halophilic mechanism and ectoine producing bacteria. Based on the phylogenetic tree of16S rRNA, polymorphism of16S-23S rRNA1SR and whole cell proteins demonstrated by a SDS-PAGE, ten Halomonas strains could be preliminarily divided into seven kinds of taxon.2) Intracellular ectoine was extract using the80%ethanol method, and quantitative detection of ectoine was performed using the high performance liquid chromatography (HPLC). The results revealed that compatible solute ectoine could be accumulated in most moderate halophiles. Five wild strains could accumulate onefold ectoine within the cells. We analyzed the accumulation of ectoine in H. ventosae QHL5by HPLC at different concentrations of sodium ions, potassium ions, magnesium ions, different pH value and temperature. The results showed that the optimal cultural conditions for ectoine accumulated in the QHL5were1.5M Na+,0.75M K+,0.2M Mg2+, and at a pH of8.0growing for35℃. Under the optimum growth salinity, the maximum concentration of ectoine was379.6mg/L, reached to167.1mg/g cell dry weight, indicating the potential application for ectoine-producing.3) The entire ectABC gene cluster (3580bp) of QHL1was cloned using genome walking. The operon was3,580bp long comprising of ectA, ectB and eclC genes, which were579bp,1,269bp and390bp respectively, and were organized in a single transcriptional unit. The intergenic regions between ectA and ectB consisted of109bp, and that between ectB and ectC was91bp. Furthermore, two putative promoters:5-dependent promoter and δ38-controlled promoter, and several conserved motifs with unknown function were identified in the upstream of the ectA gene. 4) Single ectA, ectB, ectC gene and the ectABC gene cluster were inserted into the expression plasmid pET-28a in order to generate the recombinant plasmids pET-28a-ectA/B/C and pET-2Sa.-ectABC, respectively. Heterologous expression of these proteins in E. coli BL21was confirmed by SDS-PAGE. The recombinant E.coli strain BL21(pET-28a-ectABC) displayed a higher salt tolerance than native E. coli cells but produced far less ectoine than the wild type QHL1strain, still needing further research. However, these results provide a framework for future genetic manipulation and gene regulation of ectoine biosynthesis pathways for use in industrial applications. |
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